PROJECT SUMMARY Filoviruses cause fulminant hemorrhagic fevers with a case-fatality rate of up to 90% in human outbreaks. Although filoviruses (Ebolavirus (EBOV) and Marburgvirus (MARV)) are endemic only to certain parts of central Africa and the Philippines, their extreme virulence and potential for weaponization have lead to the determination that both are high priority biothreats to US national security and medical countermeasures have been prioritized for acquisition into the Strategic National Stockpile. While strategies for passive immunotherapy have made significant progress recently, vaccination remains the most economical and technically feasible approach to protect larger groups of people from acquiring one of these viral infections. While multiple Ebola vaccine candidates have recently made significant progress on their clinical development path, it is unknown which of the filoviruses may next cause a public health emergency. Therefor a continuing need exists to develop a universally safe, multivalent filovirus vaccine that meets stockpiling requirements. The overall goal of this project is to develop a thermostable, trivalent, recombinant subunit filovirus vaccine that can protect at risk populations against infection by all pathogenic strains of EBOV (Zaire Ebolavirus), SUDV (Sudan Ebolavirus), and MARV. Thermostability will be achieved by lyophilization of the recombinant antigens allowing reconstitution at the time of use. The highly purified recombinant filovirus subunit proteins are expressed from stably transformed insect cells. A key advantage of this production system is the ability to consistently produce large quantities of pure, stable, and properly folded viral proteins. Immunoaffinity chromatography is key for the highly efficient production and is being facilitated by the use of plant-expressed monoclonal antibodies. Unlike other vaccine technologies, the recombinant subunit approach permits inclusion of antigens from diverse pathogens to achieve truly broad spectrum efficacy. Fine tuning of antigen dosing, immunization schedule, and adjuvant selection allow the rapid inclusion of new or modified targets into a core vaccine formulation. This core formulation will be further defined during the proposed work. The unique advantages of our platform are initially targeted at demonstrating feasibility in a candidate with protection against three filoviruses, having a safety profile only achievable with the use of highly purified subunit proteins. This research is divided into four Specific Aims: In Aim 1, the trivalent formulation will be tested for efficacy against each of the three filoviruses in non-human primate (NHP) models of filovirus disease. In Aim 2 the immunogenicity of lyophilized antigens will be confirmed in mice and then the lyophilized trivalent formulation will be tested against challenge with each of the viruses in comparison to a liquid (non-lyophilized) formulation using NHP models. In Aim 3, we will define the final dose level and test the durability of protection of the final formulation (up to 12 months). Aim 4 is focused on antigen production methods development establishing a fully scaleable and controlled process to generate well-characterized antigen lots of the three antigens.